Iron accumulation in the striatum predicts aging-related decline in motor function in rhesus monkeys

Changes in the nigrostriatal system may be involved with the motor abnormalities seen in aging. These perturbations include alterations in dopamine (DA) release, regulation and transport in the striatum and substantia nigra, striatal atrophy and elevated iron levels in the basal ganglia. However, the relative contribution of these changes to the motor deficits seen in aging is unclear. Thus, using the rhesus monkey as a model, the present study was designed to examine several of these key alterations in the basal ganglia in order to help elucidate the mechanisms contributing to age-related motor decline. First, 32 female rhesus monkeys ranging from 4 to 32 years old were evaluated for their motor capabilities using an automated hand-retrieval task. Second, non-invasive MRI methods were used to estimate brain composition and to indirectly measure relative iron content in the striatum and substantia nigra. Third, in vivo microdialysis was used to evaluate basal and stimulus-evoked levels of DA and its metabolites in the striatum and substantia nigra of the same monkeys. Our results demonstrated significant decreases in motor performance, decreases in striatal DA release, and increases in striatal iron levels in rhesus monkeys as they age from young adulthood. A comprehensive statistical analysis relating age, motor performance, DA release, and iron content indicated that the best predictor of decreases in motor ability, above and beyond levels of performance that could be explained by age alone, was iron accumulation in the striatum. This suggests that striatal iron levels may be a biomarker of motor dysfunction in aging; and as such, can be monitored non-invasively by longitudinal brain MRI scans. The results also suggest that treatments aimed at reducing accumulation of excess iron in the striatum during normal aging may have beneficial effects on age-related deterioration of motor performance.     

Oxidative damage and sensitivity to nociceptive stimulus and opioids in aging rats

Oxidative stress contributes to aging and may cause alterations in pain and analgesia. Knowledge about effects of oxidative stress on the opioid system is very limited. This project was designed to determine the relationship between age-related oxidative damage and opioid antinociception. Three age groups of male Fischer 344 rats were tested for pain sensitivity and responses to morphine and fentanyl using the hot plate method. Oxidative stress markers in various brain regions were measured. With advancing age, nociceptive threshold and antinociceptive effects of opioids decreased significantly. There was a significant negative correlation between morphine antinociception and protein oxidation in cortex, striatum, and midbrain (r(2)=0.73, 0.87, and 0.77, respectively), and lipid peroxidation in cerebral cortex, hippocampus, and striatum (r(2)=0.73, 0.61, and 0.71, respectively). Similar correlation was observed between oxidative stress markers and fentanyl antinociception. These findings demonstrate that the age-related increase in oxidative damage in brain is associated with a significant decrease in the antinociceptive effects of opioids.     

Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina

Heat shock proteins (HSPs) are highly conserved proteins playing a protective role under deleterious conditions caused by a wide variety of pathophysiological, including environmental stresses. Glutathione (GSH) is known to play a critical role in the cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH depletion induced cell death in the retina, but the mechanism(s) of cellular protection were not clear. Unregulated oxidative stress was induced by depletion of intracellular GSH by systematic administration of buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. After 0, 1, 4 and 7 days of BSO administration, we examined expression of both large and small HSP mRNAs (hsp90alpha, hsp90beta, hsp70, hsp60 and hsp25) in oxidative-stressed mouse retina. Of large HSPs, only hsp70 expression was significantly decreased from 1 day after BSO injection, whereas expression of other large hsps was not changed on day 1. Expression of hsp60 decreased on 4 days, whereas expression of hsp90 decreased on 7 days after BSO administration. Different from large HSPs, a small HSP, hsp25 increased its expression to a great extent from 1 day after BSO administration. Taken together, our results show that unregulated oxidative stress could induce differential expression of HSPs, which, in turn, may play distinct roles in the cellular defense. Targeting HSPs, therefore, may provide novel tools for treatment of retinal degenerative diseases such as glaucoma, retinopathy or age-related macular degeneration.     

Induction of apoptosis signal-regulating kinase 1 and oxidative stress mediate age-dependent vulnerability to 3-nitropropionic acid in the mouse striatum

The mitochondrial toxin, 3-nitropropionic acid (3-NP), produces age-dependent oxidative stress and selective striatal damage, which may simulate Huntington's disease starting in middle age. Recent reports showed that apoptosis signal-regulating kinase 1 (Ask1) activated by oxidative stress triggers a cell death signaling pathway. 3-NP was injected to the striatum in C57BL/6J mice. We have confirmed that striatal lesion volume and DNA fragmentation were age-dependent after 3-NP treatment. In the non-injured striatum of the middle-aged group, the protein levels of Ask1 and its active form, phosphorylated Ask1 (pAsk1), were significantly higher than in the young group. Ask1 increased more in the 3-NP injured striatum of the middle-aged group than in the non-injured striatum, and subsequently the activity of pAsk1 was significantly higher than in the young group. However, middle-aged SOD1Tg mice showed significant reductions of Ask1 and pAsk1 in the injured and the non-injured striatum compared to the middle-aged group. In particular, apoptosis signal transduction and cell death were significantly inhibited by the reduction of Ask1 expression using siRNA. Present results suggest that age-related upregulation of Ask1 and oxidative stress may mediate age-dependent striatal vulnerability to 3-NP.     

Role of heat shock protein 70 in induction of stress fiber formation in rat arterial endothelial cells in response to stretch stress

We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress.     

Kainate-induced seizures, oxidative stress and neuronal loss in aging rats

Aging is a significant risk factor for developing epilepsy. The mechanisms underlying age-related increase in seizure susceptibility and resultant injury remain unknown. Oxidative stress is an important mechanism that contributes to diverse age-related disorders. Whether age-related increased seizure susceptibility is accompanied by increased oxidative stress remains unknown. The goal of this study was to determine if aging per se increases the susceptibility of rats to kainate-induced behavioral seizures and oxidative stress. Adult (3-4 month-old) and aging (18-19 month-old) Sprague-Dawley rats were administered a single low dose of kainate (5 mg/kg, s.c.) or saline. Behavioral seizures were monitored in all four groups for a period for a period of approximately 6 h. Oxidative stress (8-hydroxy-2'deoxyguanosine/2-deoxyguanosine; 8OHdG/2dG) was assessed 24 h following kainate injection. Stereological assessment of cell counts was performed in hippocampal tissue 7 days following kainate injection. In adult rats, administration of the low dose of kainate did not produce significant behavioral seizures, oxidative stress or cell loss. However, aging rats exhibited intense behavioral seizures consistent with status epilepticus following the low dose of kainate. In aging rats, kainate produced a significant increase in oxidative DNA damage (8OHdG/2dG) and neuronal loss in cornu ammonis regions 3 and 1 (CA3 and CA1), but not dentate gyrus compared with both age-matched controls and adult kainate-treated rats. These data suggest that the process of aging per se increases kainate-induced seizure susceptibility, oxidative stress and hippocampal pyramidal cell loss.     

Compartmentalization of calmodulin and tubulin in the male C57BL/6J mouse brain: heterogeneity of age changes in calmodulin compartments

Calmodulin (CaM) and tubulin were analyzed by radioimmunoassay in subcellular fractions prepared from cerebral cortex and striatum of aging male C57BL/6J mice. Three fractions were prepared by a new procedure: cytosol (soluble); EGTA-releasable, membrane-bound; and detergent-extractable (Triton X-100), membrane-bound fractions. CaM concentration in all three fractions prepared from striatum showed small (10-15%, p less than 0.05) decreases with age (3-31 months). Cortical CaM concentrations were less affected by age, and only the EGTA-releasable fraction decreased. To compare functional activity and immunoreactivity of CaM, soluble CaM was also assayed by the activation of cyclic nucleotide phosphodiesterase (PDE). The radioimmunoassay and PDE activation assays gave equivalent results, suggesting that no alteration occurred with age in biological activity of CaM, via post-translational modification or other mechanisms. Soluble and particulate tubulin concentration did not change significantly with age in either brain region. The changes observed in striatal CaM, particularly in membrane-bound compartments, could contribute to the age-related decline in mammalian basal ganglial function.     

Effect of age and oxidative stress on tyrosine phosphorylation of ZAP-70.

Oxidative stress is believed to be one of the leading contributors to the aging process and loss of cellular function with aging. Although, several age-associated phenomena have been correlated with reactive oxygen species, the role of oxidative stress in the age-related decline of T-cell activity is not yet clear. The present study was carried out to determine the effect of reactive oxygen species on tyrosine phosphorylation of ZAP-70, a key enzyme in the T-cell signal transduction pathway. Our previous as well as present studies have demonstrated an age-related down-regulation of tyrosine phosphorylation of ZAP-70 following activation of freshly isolated T cells with various stimulating agents. Currently, we observe that under mild and chronic oxidative stress, tyrosine phosphorylation of ZAP-70 is impaired following stimulation of the T cells with anti-CD3 antibody. Interestingly, in contrast to our observation using freshly isolated T cells, there is no age-associated impairment of tyrosine phosphorylation of ZAP-70 when T cells, cultured for 2 days in a serum-free medium in the presence or in the absence of oxidative stress, are stimulated with anti-CD3 antibody. The current observation suggests that the age-associated down-regulation of tyrosine phosphorylation of ZAP-70 is not an intrinsic defect inherent of the T cells due to aging and is reversible. We also observed that under oxidative stress in vitro, there was no significant difference in inhibition of tyrosine phosphorylation of ZAP-70 in T cells isolated from elderly and young donors. Finally, it was also noted that the down-regulation of tyrosine phosphorylation of ZAP-70 under oxidative stress in young and elderly donors was not due to impairment in the net expression of ZAP-70 under oxidative stress thereby suggesting that dysregulation in the balance of kinase-phosphatase activities under oxidative stress might have been implicated in the observed phenomenon.     

Aged F344 rats exhibit altered electrophysiological activity in locomotor-unrelated but not locomotor-related striatal neurons

Multi-wire electrode arrays were chronically implanted and striatal electrophysiological activity was recorded in young (4-9 months) versus aged (24-29 months) Fischer 344 (F344) rats in order to determine whether locomotor-related striatal neurons exhibit age-related changes in electrophysiological activity during freely-moving conditions. Individual neurons were classified as locomotor-related if they exhibited significant differences in their firing rates between periods of locomotion versus periods of non-movement. While the activity of locomotor-related striatal neurons did not differ between young and aged rats, neurons that were not related to locomotion exhibited significantly greater activity in the aged rats during both periods of non-movement and bouts of locomotion. These results suggest that in the aged striatum, increased activity of nonlocomotor-related neurons may contribute to hypokinesia through their influence on basal ganglia output nuclei. Such studies may aid in the understanding of movement disorders seen in aging and Parkinson's disease.     

Selective alteration of mouse brain neurotransmitter release with age

The release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from various brain regions was investigated in young (3 month) and old (30 month) Balb/c mice. Aging increased the basal release of GLU (77%) and DA (29%) in striatum and GLU in hippocampus (94%); the concentrations of these neurotransmitters in the media after K+ stimulation were unaltered by aging. Although the basal release of ACh was not altered by age, K+-stimulated ACh release was reduced in striatum. The age-related increases in basal GLU and DA release may be important in the pathophysiology of cell death during aging.     

Up-regulation of inducible heat shock protein-70 expression in multiple sclerosis patients

Abstract

Inducible heat shock protein (HSP)70 (HSP70-1A and HSP70-1B proteins) is a chaperone responsible for assisting proper protein folding. Following stress conditions, HSP70 is highly up-regulated to mediate cytoprotective functions. In addition, HSP70 is able to trigger innate and adaptive immune responses that promote the immune recognition of antigens and to act as a cytokine when it is released. The data in the literature are controversial with regard to expression studies in peripheral blood mononuclear cells (PBMCs). In the present study, we aimed to examine if alterations of HSP70-1A/B expression are involved in the autoimmune pathogenesis of multiple sclerosis (MS). We determined both mRNA and protein expression in PBMCs of MS patients and healthy donors (HDs). We found a baseline increased expression of the HSPA1A gene in PBMCs from MS patients compared with HDs. Gene expression findings were associated with an increased protein expression of HSP70-1A/B in T lymphocytes (CD4+ and CD8+) and monocytes from MS patients under basal conditions that may reflect the immunological activation occurring in MS patients. We also provided evidence that heat shock (HS) stimulus induced HSP70-1A/B protein expression in HDs and MS patients, and that?HS-induced HSP70-1A/B protein expression in monocytes correlated with the number of T2 lesions at baseline in MS patients. However, after lipopolysaccharide inflammatory stimulus, monocytes from MS patients failed to induce HSP70-1A/B protein expression. Our data hint at altered immune responses in MS and may indicate either a state of chronic stress or increased vulnerability to physiological immune responses in MS patients.     

Age-related changes in expression of metallothionein-III in rat brain

Metallothionein (MT)-III is a metal binding protein, called growth inhibitory factor, and is mainly expressed in the central nervous system. Since MT-III decreases in the brain of Alzheimer's disease (AD), a growing interest has been focused on its relationship to neurodegenerative diseases. To clarify age-related changes in the MT-III expression and its inducibility against oxidative stress, we analyzed the expression of MT-III and its mRNA in the brain of lipopolysaccharide (LPS)-treated aged rats. In the frontal cortex, basal expression of MT-III mRNA was significantly increased with aging, while it was observed no induction of MT-III mRNA against LPS administration in the aged rat brain. MT-III immunopositive cells were increased in the frontal, parietal and piriform cortices, hypothalamus and amygdaloid nucleus with aging. The LPS treatment induced MT-III expression in the brain of young-adult rats, but not in the aged rat brain. Furthermore, the MT-III induction with LPS treatment was mainly observed in oligodendrocyte and microglia. In the present study, we showed that inducibility of brain MT-III against oxidative stress may be reduced with aging. Since it has been reported that MT-III has neuroprotective roles as an antioxidant, present results suggest that MT-III is closely related to the neurodegeneration in the aged animals.     

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation,thermal or oxidative stress

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.     

Blueberry supplemented diet reverses age-related decline in hippocampal HSP70 neuroprotection

Dietary supplementation with antioxidant rich foods can decrease the level of oxidative stress in brain regions and can ameliorate age-related deficits in neuronal and behavioral functions. We examined whether short-term supplementation with blueberries might enhance the brain's ability to generate a heat shock protein 70 (HSP70) mediated neuroprotective response to stress. Hippocampal (HC) regions from young and old rats fed either a control or a supplemented diet for 10 weeks were subjected to an in vitro inflammatory challenge (LPS) and then examined for levels of HSP70 at various times post LPS (30, 90 and 240 min). While baseline levels of HSP70 did not differ among the various groups compared to young control diet rats, increases in HSP70 protein levels in response to an in vitro LPS challenge were significantly less in old as compared to young control diet rats at the 30, 90 and 240 min time points. However, it appeared that the blueberry diet completely restored the HSP70 response to LPS in the old rats at the 90 and 240 min times. This suggests that a short-term blueberry (BB) intervention may result in improved HSP70-mediated protection against a number of neurodegenerative processes in the brain. Results are discussed in terms of the multiplicity of the effects of the BB supplementation which appear to range from antioxidant/anti-inflammatory activity to signaling.     

A survey of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system

Levels of the neuropeptides substance P, somatostatin, and neurotensin were measured by radioimmunoassay in regions of the rat and human central nervous system (CNS) in aging. Somatostatin levels were significantly lower only in the corpus striatum of older rats. Substance P levels and neurotensin levels were generally stable with aging as were levels of somatostatin in regions other than the corpus striatum. In post-mortem human CNS tissues, no significant negative correlations of levels of the three peptides were observed with time to refrigeration or time to freezer for the samples. In the human CNS, there were no significant age-related alterations in substance P levels in frontal cortex, thalamus, hypothalamus, caudate nucleus, globus pallidus, or substantia nigra. There was a significant age-related decrease in substance P levels in the human putamen. This age-related decrease was not present in tissues from victims of Huntington's disease nor was there any striking difference in substance P levels as a function of duration of the disease. There were no significant age-related changes in somatostatin levels in human frontal cortex, caudate nucleus, putamen, medial globus pallidus, or substantia nigra. Among these same regions, there was a significant age-related decrease in neurotensin levels only in the pars compacta and pars reticulata of the human nigra. These results implicate neuropeptides in aging processes in certain regions of the CNS. There are differences between rats and humans with respect to neuropeptides in the aging process in the CNS. Deterioration of some neuropeptide pathways in and to human basal ganglia may be involved in the suspected functional deterioration of parts of the extrapyramidal system in aging.     

Effect of Vitamin C Supplements on Antioxidant Defence and Stress Proteins in Human Lymphocytes and Skeletal Muscle

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo . Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant-induced expression of protective enzymes and HSP content. Lymphocytes from non-supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day⁻¹ for 8 weeks was found to increase the serum vitamin C concentration by ∼50 %. Lymphocytes from vitamin C-supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non-supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C-supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).     

A survey of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system

Levels of the neuropeptides substance P, somatostatin, and neurotensin were measured by radioimmunoassay in regions of the rat and human central nervous system (CNS) in aging. Somatostatin levels were significantly lower only in the corpus striatum of older rats. Substance P levels and neurotensin levels were generally stable with aging as were levels of somatostatin in regions other than the corpus striatum. In post-mortem human CNS tissues, no significant negative correlations of levels of the three peptides were observed with time to refrigeration or time to freezer for the samples. In the human CNS, there were no significant age-related alterations in substance P levels in frontal cortex, thalamus, hypothalamus, caudate nucleus, globus pallidus, or substantia nigra. There was a significant age-related decrease in substance P levels in the human putamen. This age-related decrease was not present in tissues from victims of Huntington's disease nor was there any striking difference in substance P levels as a function of duration of the disease. There were no significant age-related changes in somatostatin levels in human frontal cortex, caudate nucleus, putamen, medial globus pallidus, or substantia nigra. Among these same regions, there was a significant age-related decrease in neurotensin levels only in the pars compacta and pars reticulata of the human nigra. These results implicate neuropeptides in aging processes in certain regions of the CNS. There are differences between rats and humans with respect to neuropeptides in the aging process in the CNS. Deterioration of some neuropeptide pathways in and to human basal ganglia may be involved in the suspected functional deterioration of parts of the extrapyramidal system in aging.     

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

Brain energy metabolism is increased during postnatal development and diminished in neurodegenerative diseases linked to senescence. The objective of this study was to determine if these conditions could involve postnatal or senescence-related shifts in activity or expression of dihydrolipoamide dehydrogenase (DLDH), a key mitochondrial oxidoreductase. Rats ranging from 10 to 60 days of age were used in studies of postnatal development, whereas rats aged 5 or 30 months were used in the aging studies. The expression of DLDH was determined by Western blot analysis using anti-DLDH antibodies and DLDH diaphorase activity was measured by an in-gel activity staining method using nitroblue tetrazolium (NBT)/NADH. Activity of DLDH dehydrogenase was measured as NAD⁺ oxidation of dihydrolipoamide. When these measures were considered in separate groups of 10-, 20-, 30-, or 60-day-old rats, all three showed an increase between 10 and 20 days of age. However, dehydrogenase activity of DLDH showed a further, progressive increase from 20 days to adulthood, in the absence of any further change in DLDH expression or diaphorase activity. No age-related decline in DLDH activity or expression was evident over the period from 5 to 30 months of age. Moreover, aging did not render DLDH more susceptible to oxidative inactivation by mitochondria-generated reactive oxygen species (ROS). Taken together, results of the present study indicate that (1) brain DLDH expression and activity undergo independent postnatal maturational increases; (2) senescence does not confer any detectable change in the activity of DLDH or its susceptibility to inactivation by mitochondrial oxidative stress.